In the last decade, major strides have been made in antiviral therapies for treatment of AIDS due to HIV infection. Currently, opportunistic infections are the primary cause of suffering and death in individuals with AIDS. Many of these infections are produced by parasites that rarely affect individuals who are not immunocompromised. Unfortunately, successful combination therapies against the HIV-1 virus still leave most patients susceptible to opportunistic parasitic infections. There are few drugs available to treat these parasitic infections. The drugs that are available suffer from a lack of selectivity, resulting in host toxicity, untoward side effects, and ineffectiveness due to development of resistance. Thus there is a need to identify novel targeting strategies that may ultimately lead to therapeutics that are more selective and less toxic. This proposal outlines mechanistic and structural studies that focus on unique bifunctional enzymes present only in parasites that may serve as a potential target for the discovery of novel anti-parasitic drugs for treatment of opportunistic infections in AIDS patients. The central theme of this proposal is that an integrated mechanistic, structural and computational evaluation of the parasitic bifunctional TS-DHFR enzyme at a molecular level will help to identify novel targeting strategies for these unique bifunctional enzymes and provide the necessary proof-of-concept to establish non-active site inhibitors as an effective and novel therapeutic approach. A long-term goal of the proposed research is to take advantage of the unique differences between parasitic and human enzymes and develop novel antiparasitic drugs for the treatment of opportunistic parasitic infections. To accomplish this goal requires a multi-disciplinary approach. We have assembled a strong collaborative team with a proven track record that will have the necessary mechanistic, structural, synthetic, computational and cell biology experience to attack this problem. There are two specific aims in this proposal both focused on comprehensive studies of protozoal bifunctional TS-DHFR enzymes. PUBLIC HEALTH RELEVANCE: Opportunistic infections are the primary cause of suffering and death in individuals with AIDS. Many of these infections are produced by parasites that rarely affect individuals who are not immunocompromised. Unfortunately, successful combination therapies against the HIV-1 virus still leave most patients susceptible to opportunistic parasitic infections and new therapies are desperately needed.